As a result of numerous studies elucidating the mechanism of targeted drug delivery, various drug-carrier systems (including nanoparticle (NP) systems), that incorporate a multi-functional surface are being developed. All such systems are based on the premise that particular ligands can only be recognized by specific cell-types and not by others, thus delivering the drug payload only in the targeted tissue. This interaction of NPs with biological membrane is an important determinant in their cellular internalization, which could depend on various factors such as NP size and surface characteristics (e.g., charge, targeting ligand, etc.), as well as on the cell membrane components. Following cellular uptake, the subcellular sorting of NP into different intracellular compartments and retention could depend on their interaction with the components of endocytic machinery, cytoskeletal components and subcellular organelles. Though functionalization of NPs is increasingly explored, successful development of efficient nanocarriers is hindered by a limited understanding and lack of methodology for assessment of the nanocarrier interaction with cellular components as well as their intracellular trafficking. Consequently, it is imperative to increase understanding of the influence of surface functionalization on the NP-cell interactions and ultimately, the phenomena of cellular internalization of NPs, with a view toward improving targeted cell delivery by minimizing non-specific interactions with non-target cells and increasing the affinity of NPs toward target cells.
NPs used for drug delivery are polymeric colloidal systems (˜100 nm diameter) formulated from a FDA-approved biodegradable and biocompatible polymer, e.g. poly DL-lactide-co-glycolide (PLGA), with one or more therapeutic agent of interest loaded in or on the particles. Polymeric NPs can be formulated to incorporate various types of therapeutic agents, including low molecular weight drugs or small molecules and macromolecules such as proteins or plasmid DNA [1,2]. PLGA NPs loaded with therapeutic agents are of special interest for intracellular drug delivery owing to their biocompatibility, biodegradability and ability to sustain therapeutic drug levels for prolonged periods of time. Moreover, the duration and levels of drug released from the NPs can be easily modulated by altering formulation parameters such as drug:polymer ratio, or polymer molecular weight and composition [3].
Various techniques have been reported for preparing polymeric NPs incorporating surface-modifying agents, such as heparin, dodecylmethylammonium bromide (DMAB), DEAE-Dextran, lipofectin, and fibrinogen [4, 5]. These techniques include:                chemical coupling of the modifying agent to the surface of pre-formed NPs;        incorporation of the modifying agent within the polymer matrix of the NP, which involves dissolution of the modifying agent into the polymer solution used to form the NPs;        adsorption of the modifying agent onto the surface of pre-formed NPs.        
In the resultant NPs, the modifying agents are either present on the NP surface, due to covalent bonding via chemical coupling agent or physical adsorption, or distributed throughout the polymer matrix. In each case, the modifying agent imparts a cationic charge to the NP surface. In the case of DMAB modification, the surface charge remains cationic, regardless of the pH value of the environment to which the NPs are exposed.
The present inventors previously carried out studies which demonstrated that biodegradable PLGA NPs following cellular internalization (via endocytosis) undergo surface charge reversal (anionic to cationic) in the acidic pH of endo-lysosomes, thus facilitating their escape into the cytosolic compartment [6-8]. However, a significant fraction of NPs undergo exocytosis and only 15% of the internalized NPs escape into the cytosolic compartment. Thus, a rapid reversal of surface charge of NPs from negative to positive is considered to be the key to rapid escape of NPs from the deleterious acidic environment of the endo-lysosomes, into the cytosol. The amount of residual poly vinyl alcohol (PVA) associated with such NPs is believed to be responsible for this surface charge reversal phenomenon and, therefore, surface charge of the NPs could be altered by varying the concentration of PVA used as an emulsifier in the formulation. This belief is based on the observation that NPs with lower amount of surface associated PVA show about 3-fold higher cellular uptake in vascular smooth muscle cells (VSMCs) than the NPs with higher residual PVA [7]. Furthermore, the amount of PVA associated with the NP surface depends on the amount of PVA, the molecular weight and degree of hydroxylation of PVA used as emulsifier in the formulation [3]. Thus, the surface properties of NPs play an important role in their cellular uptake and can potentially influence the efficiency of cytosolic drug delivery.
Having shown that polymeric NPs are capable of endo-lysosomal escape due to their selective surface charge reversal in the acidic environment within endo-lysosomes, further investigation was conducted for alternative NP formulations having improved cellular uptake and more efficient cytoplasmic drug delivery.